The present invention relates to an advanced thermal gradient DNA chip (ATGC), a substrate for ATGC, a method of manufacturing of ATGC, a method and an apparatus for biochemical reaction and a storage medium.
As a method for determining a base sequence of a nucleic acid, the method for detecting hybridization between a single stranded polynucleotide of interest and a single stranded oligonucelotide probe previously designed, by using the polynucleotide detection chip with the single stranded oligonucelotide probes immobilized on its different areas depending on the type of sequences, are known. Examples of the polynuceltiode detection chips include polynucleotide detection chips for diagnosis, where DNAs complementary to specific mutated sequences of interest are arranged (Science, Vol. 270, 467-470, 1995) and those for SBH (Sequencing By Hybridization) method, in which the oligonucleotide probes capable of hybridizing with all the possible base sequences existing in a sample are provided on the chips, for determining the base sequences of the subjects of measurement (J. DNA Sequencing and Mapping, Vol.1, 375-388, 1991).
The thermal stability of hybridization between oligonucleotide probes and the single stranded polynucleotide varies depending on the types of base sequences. The reason for this is as described in the following. The bonding between adenine (A) and thymine (T) or adenine (A) and uracil (U) is of double hydrogen bond per base pair, while the bonding between guanine (G) and cytosine (C) is of triple hydrogen bond per base pair (see FIG. 11), resulting in some differences in bonding strength between these two types of bondings. Since the G-C bond is greater in strength than the A-T bond (see FIG. 12A), the thermal stability of the former is higher. Therefore, comparing the thermal stability of hybridization of sequences with equal base length, the thermal stability of hybridization involved by only A-T or A-U bond is lowest, while that involved by only Gxe2x80x94C bond is highest. In general, the thermal stability of hybridization is represented by the temperature (melting temperature, hereinafter referred to as Tm) at which both bonding and dissociation exist at rate of 50% respectively (FIG. 12B).
Taking an example of the oligoucleotide DNA probe of octamer, the Tm of the duplex DNA which consist of the A-T bondings, is 15.2xc2x0 C. (a value calculated by the % GC method (Breslauer K. J., et. al., xe2x80x9cPredicting DNA Duplex stability from the base sequencexe2x80x9d, Proc., Natl. Acad. Sci., USA83, 3746-3750), while the Tm of the duplex DNA which consist of the Gxe2x80x94C bondings, is 56.2xc2x0 C., giving a difference of 41.0xc2x0 C. (FIG. 12C).
As indicated above, when the value of Tm of the hybridization for each probe varies largely, it is necessary to carry out hybridization assay at Tm of each probe, respectively. When a temperature is higher than Tm, a single stranded polynucleotide is hard to bond effectively with a probe. On the other hand, when a temperature is lower than Tm, the background noise resulting from the mismatch bonding increases, leading to the decline of measuring resolution. Thus, in a case where different kinds of probes are immobilized on the polynucleotide detection chip, when the probes are hybridized with the single stranded polynucleotide sample while keeping the temperature constant on the chip, this gives rise to problems such as differences in the amount of the formation of hybridization and differences in mismatching probability occurring due to the difference in thermal stability among individual probes.
Conventionally, in order to resolve the above-described problems, an attempt has been made such as adjusting the salt concentration in solvent or varying the density or the base length of the probes to be immobilized on the detection chip for each probe, while keeping the temperature equal for hybridization for all the probes on the detection chip. Such an attempt, however, is not sufficient for fully eliminating the effect of the difference in Tm.
As an example of the means for resolving this problem, there is Laid Open Japanese Patent No. H11-127900 disclosing a method wherein conductive heating track is provided around each analytical electrode or a method wherein each analytical electrode is heated by means of laser. However, the Laid Open Japanese Patent No. H11-127900 discloses a method characterized by only heating the analytical electrode and no means controlling, for example, the temperature of the analytical electrode to a constant level, are disclosed.
An object of the present invention is to provide a biochemical reaction detection chip and its substrate capable of controlling the temperature for biochemical reaction including hybridization of the oligonucleotide probe with polynucleotide.
Another object of the present invention is to provide a apparatus and a method for enabling the biochemical reactions in a plurality of reaction systems to progress simultaneously at temperatures controlled for individual reaction systems and an associated data storage medium.
Further another object of the present invention is to provide a substrate of a biochemical reaction detection chip comprising a plurality of islands of a heat conductive material formed on a membrane, the islands being placed apart from each other and each island being provided with a temperature controller.
It is preferable for the membrane to be formed from a material having a high insulating ability, heat insulating ability and physical strength. The electric conductivity of 108xcexa9xc2x7m or more is sufficient for the membrane material, preferably, 1010xcexa9xc2x7m or more. The heat conductivity of 10 w/mk or less is sufficient for the membrane material, preferably, 1 w/mk or less.
It is easier to control the temperature of each island by forming the membrane from a material having a high (electrical) insulating ability and a high heat insulating ability. The membrane may be formed, for example, from at least one of a group of materials such as silicon nitride, silicon oxide, aluminum oxide, Ta2O5, or may be a composite membrane of these materials. Among these, the composite membrane of SiN and SiO2 is preferable. Since SiN has resistance to alkali, probes can be immobilized on SiN membrane by means of silane coupling in alkali solution. Further, the SiN membrane is capable of protecting the electronic circuit for temperature control provided thereunder from the solution such as sample solution.
The film thickness of 1-500 xcexcm is sufficient, preferably, 5-20 xcexcm.
It is preferable to make an indent for the area for fixing the probe of the membrane. Such indent is convenient for holding the sample solution on a chip when letting the biochemical reaction take place by bringing sample solution into contact with the probe.
Further, a resist membrane may be formed on the surface opposite to the islands. The resist membrane may be of a photosensitive polyimide resin or the like.
A plurality of islands of a heat conductor are formed on the membrane. xe2x80x9cA plurality of islandsxe2x80x9d means at least 2 islands, preferably 10-1000 islands, although the number of the islands is not defined. A plurality of islands may be arranged either in line or 2-dimensionally, that is, in a first direction (row) and a second direction (column).
The islands are formed from a heat conductor. Examples of heat conductors include crystals of Si, metals such as Ag, Au, Cu and silicones such as polysilicone and amorphous silicone. The heat conductor constituting the islands is preferable to be electrically insulatable from the temperature controller. Silicone is preferable as a heat conductor to form the islands, since it is a good heat conductor and can be electrically insulated from the temperature controller. The insulation between the heat conductor and the temperature controller can be secured by forming a pn junction in the silicone.
The islands are spaced from each other. The spaces among the islands serve as a substitute for heat insulating material, and so the temperature of each island can easily be controlled independently.
The size of 10-1000 xcexcm2 is sufficient for an island, preferably 50-500 xcexcm2. The interval of 50-1000 xcexcm between islands is sufficient, preferably 100-500 xcexcm. The shape of islands are not defined specifically. For instance, when forming the islands of Si crystal from a flat sheet of Si crystal having 100 planes as a surface by removing unnecessary portion by etching with KOH, 111 planes are exposed during manufacturing process, making a regular pyramid-like form.
Each of a plurality of islands is provided with the temperature controller. More particularly, it is preferable to provide a heating circuit and a temperature detection circuit for each island. The heating circuits and the temperature detection circuits may be controlled to operate independently either for each island or for each group of islands.
Further, where a plurality of islands are arranged two-dimensionally, the heating circuits and the temperature detection circuits may be controlled to operate independently for each (first or second) line. The size of the biochemical reaction detection chip is sufficient to be 25 mm2-100 cm2, preferably 100 mm2-14 cm2.
With a biochemical reaction detection chip manufactured by immobilizing probes on a substrate of the biochemical reaction detection chip according to the present invention, the influence of the temperature of an adjacent probe cell (reaction system) can be reduced so that the biochemical reaction is allowed to progress at a proper temperature on each of the probe cells (reaction system).
The substrate for the biochemical reaction detection chip according to the present invention is preferable to be provided with heat sinks for allowing heat to escape outside installed among the islands. Each heat sink is preferable to have a structure (e.g., a mesh structure) that prevents it from directly contacting with islands. The heat sinks may be installed either only for one direction or both directions of first and second directions. Where the probes are divided into groups according the proximity of optimal temperatures of biochemical reactions and fixed on the membrane, heat sinks may be provided for each area of such groups, respectively.
It is preferable to form heat sinks from materials having good heat conductivity, such as Si, Au, Ag, Cu and the like.
Forming heat sinks among the islands enables heat to escape outside before being transmitted from any adjacent islands.
The distance between an island and a heat sink is sufficient to be 10-500 xcexcm, preferably 10-250 xcexcm.
Further, the present invention relates to a method for manufacturing the substrate for the biochemical reaction detection chip, more particularly to a method comprising the steps of:
(a) forming a membrane on one surface of a flat sheet of heat conductor, and
(b) forming islands of heat conductor by removing unnecessary portion from the other side of the flat sheet of heat conductor.
In the method mentioned above, temperature controller may be provided on one surface of the flat sheet of heat conductor, and the membrane may be formed thereon.
As one embodiment of the manufacturing method for the substrate of the biochemical reaction detection chip, a mask having a desired pattern may be provided on the surface of the flat sheet of heat conductor opposite to the surface the membrane is formed so that the masked surface can be etched until the membrane formed on the other surface comes to be exposed to form islands of heat conductor on the membrane corresponding to the pattern of the masking. The mask, for example, may be of a silicon nitride membrane.
Further, the present invention provides a biochemical reaction detection chip with a probe immobilized on the substrate of the biochemical reaction detection chip described above.
The surface of silicon nitride membrane is preferable for having the probes immobilized thereon. In this case, the probe with amino group can be immobilized on the surface of silanized silicon nitride membrane by means of silane coupling.
xe2x80x9cProbexe2x80x9d means substances which can specifically detect a particular substance, site, state and the like, and includes oligonucleotide DNA/RNA probes, protein probes such as antibodies, and the like. In the case of oligonucleotide DNA/RNA probe, the number of bases is sufficient to be 4-500 nt (nucleotide), preferably 8-200 nt (nucleotide). The oligonucleotide probe may be either of single strand or double strand, preferably of single strand from the point of the efficiency of the bonding between the probe and the subject.
Probes can be immobilized on the membrane on the substrate of the biochemical reaction detection chip by a known method. For instance, when the probe cells on the membrane are silanized, a probe with amino group can be immobilized on the membrane by silane coupling. The islands should be provided under the probe cells on membrane.
Further, after the probe is immobilized, the area of the membrane other than that of the probe cell is preferable to be coated with polylysine to make inactive the binding site which is not binding to the probe of the silane coated surface. Polylysine coating can prevent sample DNA, RNA and the like from binding non-specifically with the silane coated surface.
The kinds of probes are not limited, and one or more kinds of probes may be used. When a plurarity kinds of probes are immobilized on a single chip, a plurality subjects of detection in one sample can be detected simultaneously. Alternatively, when many kinds of probes are immobilized on a single chip, one kind subject of detection in a plurality of samples can be detected simultaneously.
The detection chip according to the present invention can be used for detecting biochemical reactions, for example, for detecting DNA, cDNA, RNA and protein, and antigen-antibody reaction.
When the biochemical reaction detection chip according to the present invention is used, the biochemical reaction can be carried out at an optimal temperature on each probe cell (reaction system) by reducing the influence of the temperature of adjacent probe cell (reaction system).
Further, the present invention provides a biochemical reaction apparatus for enabling the biochemical reactions in a plurality of reaction systems to take place on a biochemical reaction detection chip, the apparatus comprising a heater for heating the whole biochemical reaction detection chip to a temperature higher than the optimal temperature for each biochemical reaction and a temperature controller for controlling the temperature of each reaction system to a temperature suitable for each biochemical reaction.
The temperature controller is preferable to control the temperature of each reaction system by minutes.
Further, the present invention provides a computer-readable strage medium storing a program for operating a biochemical reaction apparatus for enabling the biochemical reactions in a plurality of reaction systems to take place on a biochemical reaction detection chip, the apparatus comprising a heater for heating the whole biochemical reaction detection chip to a temperature higher than the optimal temperature for each biochemical reaction, and a temperature controller for controlling the temperature of each reaction system to a temperature suited for each biochemical reaction.
The present invention also provides the reaction methods given below.
(1) A method for carrying out biochemical reactions in a plurality of reaction systems simultaneously at temperatures controlled respectively for each reaction system, comprising the steps of:
(a) heating all the reaction systems to a temperature higher than the optimal temperature for the biochemical reaction in each reaction system, and
(b) lowering the temperature of each reaction system to an optimal temperature for each biochemical reaction in each reaction system and maintaining the temperature for a certain period of time.
(2) A method according to (1) above, wherein the heating process (a) is carried out in an incubator.
(3) A method according to (1) above, wherein the process for lowering the temperature (b) is carried out by stopping heating process (a) or using a cooler.
(4) A method according to (1) above, wherein the biochemical reaction is the hybridization between polynucleotide and oligonucleotide, and the optimal temperature for the biochemical reaction is the melting temperature of double strand formed with the oligonucleotide and its complementary strand.
(5) A method according to (4) above, wherein the polynucleotide is DNA in a sample, and the oligonucleotide is oligonucleotide probe of the biochemical reaction detection chip.
Further, the present invention also provides a storage medium storing a program for performing the biochemical reaction controlled by a computer.
(6) A computer-readable strage medium storing a program for executing a method for performing a plurarity of biochemical reactions in a plurarity of reaction systems simultaneously while controlling the temperature for each reaction system, the method comprising the steps of:
(a) heating all the reaction systems to a temperature higher than the optimal temperature for the biochemical reaction in each reaction system, and
(b) lowering the temperature of each reaction system to an optimal temperature for each biochemical reaction in each reaction system and maintaining the temperature for a certain period of time.
When the optimal temperature for a biochemical reaction is the melting temperature of double strand formed with oligonucleodide probe and its complementary strand, the temperature higher than the optimal temperature for the biochemical reaction is preferably a temperature at which the double stranded nucletiode dissociates completely, for example, a temperature between 90xc2x0 C.-99xc2x0 C. The temperature suitable for the biochemical reaction may be a temperature around the melting temperature, e.g., within the melting temperature xc2x12xc2x0 C.
An embodiment of the present invention will be described in the following. A sample is injected into reaction systems on a biochemical reaction detection chip. Then, the chip is covered and placed in an incubator and heated to a maximum temperature, e.g., 90xc2x0 C. Normally, the incubator is provided with a heater and a cooler so that the internal temperature can be adjusted to a predetermined temperature. The temperature of the incubator is then set to a minimum temperature, e.g., 15xc2x0 C., to bring down the temperatures of all the reaction systems. When the temperature of each reaction system (e.g., a probe cell) is become lower than a set temperature (e.g., a melting temperature of double strand formed with each probe and its complementary strand), the heater is turned on to proceed the biochemical reaction while maintaining the set temperature for each reaction system for a period of time (e.g., 12 hours). After the reaction, the reaction system (e.g., the probe cell) is washed, and the biochemical reaction is detected to process the data obtained as a result of the detection.
For detection, a fluorescent marker is generally bound to a sample so that the amount of fluorescence of the marker bound to the probe can be measured with a co-focal-point microscope, and the amount of the bonded sample is calculated on the basis of the amount of the fluorescence.
Normally, biochemical reactions started at an optimal temperature for the biochemical reactions only by heating the reaction systems. However, this method frequently results in the probe binding with a substance other the subject that to be detected, causing the noise in detecting the subject. Therefore, raising the temperature of reaction systems to a level higher than the optimal temperature for the biochemical reactions and then lowering to the optimal temperature can reduce the probability that the probe is bound with a substance other than the subject of detection, thereby reducing the noise in detecting the subject.
For the hybridization of the oligonucleotide probe and polynucleotide, the optimal temperature is the melting temperature of the double strand formed with the probe and its complementary strand. When the reaction is allowed to proceed at the melting temperature only by heating the reaction system, the oligonucleotide probe may bind with a nucleotide other than the nucleotide having the complementary strand to the probe (the subject of detection), resulting in so-called mismatching that causes the noise in detecting the subject. However, when the temperature of the reaction system is once raised to a level higher than the melting temperature, and then lowered to the melting temperature, the probability that the probe is bound with a nucleotide other than the nucleotide having the complementary strand to the probe is reduced, thereby contributing to the decrease of the noise in detecting the subject. In the method according to the present invention, the temperatures of all the reaction systems are first raised to the levels higher than the optimal temperatures for reactions and then lowered to the optimal temperatures, and maintained the temperatures for a certain period of time, for example, by supplying necessary amount of heat to the reaction system by heaters.
Consequently, comparing with the method in which the temperature of each reaction system is raised to its optimal temperature and the reaction is proceeded while maintaining the temperature, the present method not only reduces the total amount of the heat to be supplied to the reaction system but also controls the temperature for each reaction system more easily. The method according to the present invention is advantageous over the conventional method in performing a number of biochemical reactions in pararell-proceeding.
Alternatively, optimal temperature for one specific reaction can be determined when the method described above is applied to a plurality of reaction systems performing the same reaction at temperatures varying from system to system.
This specification includes part or all of the contents as disclosed in the specification and/or drawings of Japanese Patent Application No.356433/1999, which is a priority document of the present application.